| Objective:In view of traditional Chinese medicine, "correlation of formula and syndrome" and biological basis of it both are the important problems in the formula. And syndrome model is the key tool in research of "correlation of formula and syndrome". At present, because the connotation of the syndrome is short of determinacy, people attach importance to disease-syndrome model, which is based on the background of the disease and has the clear physiopathology. However, the evaluation of disease-syndrome model only remains in researches of behavioristics of syndrome and systemic physiopathology, but there is few research of molecular level (e.g. proteome).In this study, based on previous studies, disease-syndrome rat models of T2DM were respectively induced by HFD or STZ/HFD, and then evaluated and compared them using proteome labled by iTRAQ. In view of "behavioristics of syndrome-systemic physiopathology-proteome", difference of their biology was stated and underlying biomarkers of T2DM and deficiency of qi and yin combined with phlegm and blood stasis syndrome were found by proteome. Moreover, effect of reinforcing qi, replenishing yin, resolving phlegm, and eliminating blood stasis formula on deficiency of qi and yin combined with phlegm and blood stasis syndrome was observed. In addition, therapeutic targets of reinforcing qi, replenishing yin, resolving phlegm, and eliminating blood stasis formula were found and the theory of "correlation of formula and syndrome" was confirmed by molecular level.Methods:1. Evaluation of models:The 35 male SD rat was randomly divided into three groups:normal group, HFD group, STZ/HFD group. Rats in the normal group were fed with basal diet, while rats in HFD and STZ/HFD group were fed with HFD for 12 weeks. In the 36th days, rats in the model group were injected with 30mg/kg STZ, T2DM was verified 72 h later by evaluating blood glucose levels with the use of blood glucose test strip. T2DM was induced successfully when the fasting blood glucose (FBG) level of rats was higher than 11.1 mmol/L. At 12 weeks, behavioristics of syndrome (fur scores, behavior scores, emotion scores, tongue texture scores, tail scores, body weight, food intake, water intake, stool weight, urine volume, grip strength) were observed and recorded. Rats were sacrificed under light ether anesthesia, then blood samples were collected from the aortaventralis and tissues (liver, skeletal muscle, adipose) were quickly collected. (1) Indicators of laboratory:Glycosylated hemoglobin (GHbA1c), fast serum insulin (Fins), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), triglyceride (TG), total cholesterin (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), thromboxane B2 (TXB2),6-ketone prostaglandin Fla (6-keto PGF1α), endothelin-1 (ET-1), glucose transporter-2 of liver (GLUT-2 of liver), glucose transporter-4 of skeletal muscle (GLUT-4 of skeletal muscle), glucose transporter-4 of adipose (GLUT-4 of adipose). (2) Serum proteome.2. Evaluation of therapeutical effects of drugs and exploration of therapy targets:The 75 male SD rat was randomly divided into two groups:normal group (n=10), model group (n=65). Rats in the normal group were fed with basal diet, while rats in model group were fed with HFD for 12 weeks. In the 36th days, rats in the model group were injected with 30mg/kg STZ, T2DM was verified 72 h later by evaluating blood glucose levels with the use of blood glucose test strip. T2DM was induced successfully when the fasting blood glucose (FBG) level of rats was higher than 11.1 mmol/L. T2DM rats were divided into three groups: STZ/HFD group, pioglitazone hydrochloride group (PIO group,2.7mg·kg·-1·-1), reinforcing qi and replenishing yin formula group (CHF(A) group,3.37g crude drug·kg-1·d-1), reinforcing qi, replenishing yin, and resolving phlegm formula group (CHF(B) group,3.84g crude drug·kg-1·M-1), reinforcing qi, replenishing yin, resolving phlegm, and eliminating blood stasis formula group (CHF(C) group,4.80g crude drug·kg-1·d-1). All rats were gavage for 14 days. At 12 weeks, behavioristics of syndrome (fur scores, behavior scores, emotion scores, tongue texture scores, tail scores, body weight, food intake, water intake, stool weight, urine volume, grip strength) were observed and recorded. Rats were sacrificed under light ether anesthesia, then blood samples were collected from the aortaventralis and tissues (liver, skeletal muscle, adipose) were quickly collected. Indicators of the laboratory in this study were the same with the above. After T2DM deficiency of qi and yin combined with phlegm and blood stasis syndrome rats were treated with CHF(C) or PIO, serum proteome was detected.Results:1. Evaluation of models:(1) HFD group:alopecia and greasy, decreased activity, somnolent, pale tongue, damp or dark red tail, polydipsia, polyphagia, diuresis, more stool, and increase of FBG and cGMP levels, as well as decrease of grip strength, LSI, GLUT-2 of liver, GLUT-4 of skeletal muscle, GLUT-4 of adipose, cAMP/cGMP, HDL-C,6-keto PGF1α levels; compared with the normal group, there were 101 differentially expressed proteins (55 up-regulation,46 down-regulation) as well as 153 biological pathways,11 signaling pathways, and 2 networks. (2) STZ/HFD group:alopecia and greasy, huddling, dispirited, ecchymosis of tongue texture and tail, polydipsia, polyphagia, diuresis, more stool, emaciation, and increase of FBG, HOMA-IR, Fins, GHbAlc, cGMP, TC, LDL-C, TXB2/6-keto PGFla levels, as well as decrease of grip strength, ISI, GLUT-2 of liver, GLUT-4 of skeletal muscle, GLUT-4 of adipose, cAMP, cAMP/cGMP, HDL-C,6-keto PGFla levels; compared with the normal group,48 differentially expressed proteins (40 up-regulation,8 down-regulation) as well as 20 biological pathways,4 signaling pathways, and 2 networks; compared with the normal group, increases of serum FCN2, apoC4, apoB100 and CYR61 levels were found by ELISA. (3) Compared with HFD group, there was 97 differentially expressed and 32 similarly expressed proteins, as well as 116 biological pathways,13 signaling pathways,2 networks in STZ/HFD group; compared with HFD group, there were decrease of serum a 113 level and increases of serum HSP71, FXII, PPKARIA, FCN2 levels in STZ/HFD group; moreover, there was no difference of serum UN13D and LBP levels between STZ/HFD and HFD group.2. Evaluation of therapeutical effects of drugs and exploration of therapy targets:(1) CHF(C) group:amendatory effects on fur scores, behavior scores, emotion scores, tongue texture scores, tail scores and increase of food intake, body weight, grip strength, ISI, GLUT-2 of liver, GLUT-4 of skeletal muscle, GLUT-4 of adipose, cAMP, cAMP/cGMP, 6-keto PGFla levels, as well as decrease of water intake, stool weight, FBG, GHbAlc, Fins, HOMA-IR, cGMP, TG, TC, LDL-C, TXB2, TXB2/6-keto PGFla, ET-1 levels were observed; there were 23 differentially expressed proteins (5 up-regulation,18 down-regulation) and 7 biological pathways after CHF(C) treatment; Increase of serum CDC42, RhoA, CYR61 levels were found in STZ/HFD group, while decreases of them were found in the CHF(C) group. (2) PIO group:amendatory effects on fur scores, behavior scores, emotion scores, tongue texture scores, and increase of food intake, urine volume, body weight, grip strength, ISI, GLUT-2 of liver, GLUT-4 of skeletal muscle, GLUT-4 of adipose, cAMP,6-keto PGF1α levels, as well as decrease of water intake, stool weight, FBG, GHbAlc, Fins, HOMA-IR, cGMP, TG, TC, LDL-C, TXB2, TXB2/6-keto PGFla, ET-1 levels were observed; there were 12 differentially expressed proteins (1 up-regulation,11 down-regulation) after CHF(C) treatment. (3) there were 8 similar targets between CHF(C) and PIO groups.Conclusions:1. Evaluation of models:(1) At 12 weeks, T2DM rat model was successfully replicated by HFD fed or HFD fed with STZ injection. The early stage of T2DM rat model induced by HFD and deficiency of qi and yin syndrome were observed, while T2DM rat model induced by HFD fed with STZ injection squinted towards deficiency of qi and yin combined with phlegm and blood stasis syndrome. (2) In view of insulin secretion, blood coagulation, and glycolysis, mechanism of disease-syndrome T2DM rat model was different. In addition, inflammation injury was common mechanism, however, inducement of inflammation injury was different. (3) FCN2, apoC4 and apoB100 were underlying biomarkers of T2DM, while CYR61 was underlying biomarker of deficiency of qi and yin combined with phlegm and blood stasis syndrome.2. Evaluation of therapeutical effects of drugs and exploration of therapy targets:(1) After CHF(C) and PIO treatment, differently therapy effects on T2DM deficiency of qi and yin combined with phlegm and blood stasis syndrome were found. (2) The mechanism of CHF(C) on T2DM deficiency of qi and yin combined with phlegm and blood stasis syndrome rats was regulation of serum proteins related with inflammation, signal transduction, protein kinase activity, and so on. (3) CDC42, RhoA, CYR61 were one of possible targets of CHF(C).In conclusion, deficiency of qi and yin combined with phlegm and blood stasis syndrome were observed in T2DM rats induced by HFD and STZ. Moreover, FCN2, apoC4 and apoB100 were underlying biomarkers of T2DM, CYR61 was underlying biomarker of deficiency of qi and yin combined with phlegm and blood stasis syndrome. In addition, therapy effects of CHF(C) on T2DM deficiency of qi and yin combined with phlegm and blood stasis syndrome were found and CDC42, RhoA, CYR61 were one of possible targets of CHF(C).
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